The present invention relates to anion exchange separation processes and novel polymers for use in such processes. It relates especially to processes in which anionic components such as anticoagulants are removed from blood.
In WO-A-93/01221 we describe various polymers and their use to coat surfaces to improve their biocompatibility. The polymers include zwitterionic groups and pendant groups which are capable of providing stable surface binding of the polymer to underlying substrate surfaces. The binding may be by provision of pendant hydrophobic groups which physisorb onto hydrophobic substrates, by counterionic attraction between pendant ionic groups on the polymer and oppositely charged groups at the substrate surface, by providing covalent attachment between coreactive pendant groups on the polymer and groups at the substrate surface or by crosslinking the polymer after coating. Post coating crosslinking may also be used to improve the stability of a polymer which is physisorbed, covalently bonded or counterionically bonded to the surface. The polymers have good hemocompatibility as indicated by the low platelet adhesion values reported in that specification.
It has also been shown that zwitterionic groups at substrate surfaces, for instance of contact lenses, show lower rates of deposition of proteins and lipids from biological liquids such as tear film. In WO-A-92/07885, reduced levels of protein deposition are described for contact lenses formed from a hydrogel of a crosslinked copolymer of copolymerisable zwitterionic monomer and non ionic comonomer.
In WO-A-93/21970 it is disclosed that microorganisms, especially bacteria, adhere to surfaces having pendant phosphoryl choline groups less than to similar surfaces without such groups present.
Another way of reducing the thrombogenicity of surfaces has involved attachment or adsorption of anti-thrombogenic active compounds to substrate surfaces. For instance heparin may be attached through covalent or counterionic bonding to surfaces. In U.S. Pat. No. 3,634,123 the binding of heparin to a surface was increased by incorporation of cationic surfactant. A related process is described in EP-A-0350161, in which a surface is first coated with a cationic surfactant and subsequently with heparin. In EP-A-0086187 the surface is first coated with a cationic polymer and subsequently with heparin. In JP-A-53/137268 a cross-linked acrylic copolymer of a cationic monomer and a polyethyleneglycol monomer is blended with polyurethane and made into tubing which can be coated with heparin. In EP-A-0086186 heparin is attached to an underlying surface through a covalent bond via the end carbohydrate unit. In U.S. Pat. No. 5,342,621, a complex of heparin with phosphatidyl choline and admixed with a polymer of caprolactone or L-lactic acid (both of which have no overall charge) and subsequently used to coat medical devices.
Generally patients who are undergoing complex operations requiring that their blood be directed through extra corporeal circuitry, require administration of heparin into the circulation to prevent the blood clotting. Subsequently the heparin has to be neutralised or removed from the blood stream. In order to remove heparin from the circulation without administering a further active compound to neutralise the heparin, it has been suggested to immobilise protamine, a cationic polypeptide used to neutralise heparin, at the surfaces of a filter used in an extra corporeal blood circuit, to scavenge heparin from a patient who has been systemically heparinised.
In J. Chromatography A (1996) 722, 87-96 and Int. Symp. Chromatog. 35th Anniv. Res. Group Liq. Chrom. Jpn 1995, 593-597, Yang et al describe ion exchange stationary phases for HPLC. The materials were based on silica to which organic groups including secondary amine groups were attached which in turn were partially derivatised to zwitterionic groups.
In a new ion exchange process according to the invention a substrate has at its surface zwitterionic pendant groups and cationic pendant groups having anionic counterions and is contacted with an aqueous solution having suspended or dissolved therein an anionically charged compound, whereby the anionic compound is ion exchanged with the counterions.
The process is of particular value for treatment of blood, especially for scavenging clotting inhibitors, for instance anionic mucopolysaccharides. The anionically charged mucopolysaccharide may be heparin or a similar is anti-thrombogenic compound such as hirudin or chondroitin sulphate, or may be alginate or hyaluronic acid. The provision of zwitterionic groups seems to minimise adsorption of other components from blood or biological fluids contacted with the substrate surface, thereby preventing fouling of the surface which may prevent ion exchange taking place. In the process of the invention the zwitterionic group, hereinafter referred to as a group X, preferably has a permanent cation, that is a quaternary ammonium or phosphonium or a tertiary sulphonium group. The cationic group at the surface, similarly, is preferably permanently cationic and thus not pH sensitive. The cationic group is preferably a group N+R53, P+R53 or S+R52 in which the groups R5 are the same or different and are each C1-4-alkyl or aryl (preferably phenyl) or two of the groups R5 together with the heteroatom to which they are attached from a saturated or unsaturated heterocyclic ring containing from 5 to 7 atoms, preferably the cationic group is N+R53 in which each R5 is C1-4-alkyl, preferably methyl.
It is preferred for the anion of the zwitterion to be a phosphate or a phosphonate group, usually a phosphate ester and is most preferably a phosphate diester and thus having a single negative charge.
Most preferably X is a group of formula 
in which the moieties X1 and X2, which are the same or different, are xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94 or a valence bond, preferably xe2x80x94Oxe2x80x94, and W+ is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C1-12-alkylene group.
Preferably W contains as cationic group an ammonium group, more preferably a quaternary ammonium group.
The group W+ may for example be a group of formula xe2x80x94W1xe2x80x94N+R233, xe2x80x94W1xe2x80x94P+R23a3, xe2x80x94W1xe2x80x94S+R23a2 or xe2x80x94W1xe2x80x94Het+ in which:
W1 is alkylene of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group W1 optionally contains one or more fluorine substituents and/or one or more functional groups; and either
the groups R23 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl or two of the groups R23 together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R23 together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R23 is substituted by a hydrophilic functional group, and the groups R23a are the same or different and each is R23 or a group OR23, where R23 is as defined above; or
Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine.
Preferably W1 is a straight-chain alkylene group, most preferably 1,2-ethylene.
Preferred groups X of the formula VI are groups of formula VA.
The groups of formula (VA) are: 
where the groups R12 are the same or different and each is hydrogen or C1-4 alkyl, and e is from 1 to 6, preferably 2 to 4.
Preferably the groups R12 are the same. It is also preferable that at least one of the groups R12 is methyl, and more preferable that the groups R12 are all methyl.
Preferably e is 2 or 3, more preferably 2. When X is a group of formula (VA) preferably B is a group of formula xe2x80x94(CR132)xe2x80x94 or xe2x80x94(CR132)2xe2x80x94, eg. xe2x80x94(CH2)xe2x80x94 or xe2x80x94(CH2CH2)xe2x80x94.
The cationic group is preferably a group N+R53, P+R53 or S+R52 
in which the groups R5 are the same or different and are each C1-4-alkyl or aryl (preferably phenyl) or two of the groups R5 together with the heteroatom to which they are attached from a saturated or unsaturated heterocyclic ring containing from 5 to 7 atoms. Preferably Q1 is N+R53 in which each R5 is C1-4-alkyl, preferably methyl.
The counterion is a suitable ion, preferably derived from a strong acid, most preferably an acid having a pKa less than 1 for instance less than 0, more preferably less than xe2x88x921 for instance an inorganic acid. Preferably the counterion is a halide, most preferably chloride.
In the ion exchange process the surface has cationic and zwitterionic groups immobilised at the surface of a substrate which is usually in particulate or membrane form. Membranes may be made of regenerated cellulose in hollow fiber form. Such fibers may be provided with the desired pendant groups by coating with a preformed polymer containing both cationic and zwitterionic groups, which can be crosslinked after coating using crosslinkable monomers as described below. Alternatively cationic and zwitterionic monomers may be graft polymerised directly onto the surface of the cellulose fibers using the process described in U.S. Pat. No. 5,453,467 using suitable monomers, of the general type described below. Alternatively such monomers could be graft polymerised onto soluble cellulose which is subsequently coated onto the fibers using the general technique described in WO-A-93/15775. Terpolymers described below can be used successfully to coat various substrates including polyesters, polycarbonates, polypropylene, polyvinyl chloride and steel and filters may include coated surfaces of any of these materials. Alternatively the polymer could be formed in the presence of a crosslinking monomer having two or more ethylenically unsaturated groups to form a crosslinked material which can be used in bulk as the ion exchange material.
The substrate may be any of those used for ion exchange, for instance based on cross-linked acrylic polymers or polystyrene based polymers or may be based on silica supports. Such substrates with suitable reactive groups, such as amine, carboxylate or hydroxyl groups are available. Alternatively they can be generated; for instance a silica substrate can be reacted with a silane such as a trimethoxy silane having an amine, chlorobenzyl or glycidoxy substituent. Reagents for attaching cationic groups to surface hydroxyl, amine or carboxylate groups are described in our earlier publication WO-A-9106020 while reagents for attaching zwitterionic groups are described in our earlier publications WO-A-9113639 and WO-A-9207858. Crosslinked polymers including zwitterionic monomers can be made as described in WO-A-9207885.
The preferred approach is to coat a particulate or membrane substrate with a preformed polymer having pendant cationic and zwitterionic groups.
The polymer having pendant zwitterionic and cationic groups is generally a copolymer of copolymerisable monomers. Whilst it is most convenient for the polymer to be formed by addition polymerisation of ethylenically unsaturated monomers, it may alternatively be a condensation polymer, or an alternative type of addition polymer, for instance formed by ring opening cyclic monomers.
Copolymers of ethylenically unsaturated monomers may be formed from monomers including
a) a zwitterionic monomer of the formula I
YBXxe2x80x83xe2x80x83I
wherein B is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally include one or more fluorine substituents;
X is an organic group having a zwitterionic moiety preferably as described above; and
Y is an ethylenically unsaturated polymerisable group; and
b) a cationic monomer of the formula II
Y1B1Q1xe2x80x83xe2x80x83II
wherein B1 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally includes one or more fluorine substituents;
Y1 is an ethylenically unsaturated polymerisable group; and
Q1 is an organic group having a cationic moiety, preferably as described hereinbefore.
Preferably a copolymer includes additional pendant groups capable of providing stable bonding at the substrate surface. Such groups are generally introduced by incorporation of termonomers into the polymerisation. A termonomer may, for instance, include a hydrophobic group which provides for physisorption at the surface, where the substrate surface is hydrophobic, or may comprise a covalent reactive group which is capable of forming a covalent bond with coreactive groups at the substrate surface. Alternatively the copolymer may be crosslinked after coating by subjecting a polymer having pendant crosslinkable groups to conditions such that crosslinking takes place.
A termonomer which has a hydrophobic group is generally of the formula III
Y2B2Q2xe2x80x83xe2x80x83III
wherein B2 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which may optionally include one or more fluorine substituents;
Y2 is an ethylenically unsaturated polymerisable group; and
Q2 is an organic group having a hydrophobic group selected from alkyl groups having at least six carbon atoms, fluorine substituted alkyl groups and alkyl groups having at least one siloxane substituent.
A covalent reactive termonomer may have the general formula IV:
Y3B3Q3xe2x80x83xe2x80x83IV
wherein B3 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally includes one or more fluorine substituents;
Y3 is an ethylenically unsaturated polymerisable group; and
Q3 is an organic group having a reactive group capable, on imposition of an external stimulus, of reacting with a coreactive group on the surface of a substrate or which is pendant on the polymer.
Reactive groups Q3 may also provide crosslinkability on the polymer. For instance such groups may react with each other or may react with different coreactive groups as pendant groups on the copolymer, for instance amine or, more usually, hydroxyl groups. Examples of reactive groups capable of crosslinking with such pendant groups or of reacting to provide covalent binding to a surface, for example an aldehyde group or a silane or siloxane group containing one or more reactive substituents such as halogen, for example chlorine, or alkoxy, generally containing from 1 to 4 carbon atoms, for example methoxy or ethoxy, or, more preferably, Q3 is a hydroxyl, amino, carboxyl, epoxy, xe2x80x94CHOHCH2Hal, (in which Hal is a halogen atom such as chlorine, bromine or iodine) succinimido, tosylate, triflate, imidazole carbonyl-amino or optionally substituted triazine group.
Preferred reactive comonomers IV which are used to crosslink the comonomer, rather than provide covalent binding to the surface, are those Q3 contains a crosslinkable cinnamyl, epoxy, xe2x80x94CHOHCH2Hal (in which Hal is a halogen atom), methylol, silyl, an ethylenically unsaturated crosslinkable group, such as an acetylenic, diacetylenic, vinylic or divinylic group, or an acetoacetoxy or chloroalkyl sulfone, preferably chloroethyl sulphone, group.
In each of the monomers I to IV the ethylenically unsaturated group is preferably selected from 
CH2xe2x95x90C(R)xe2x80x94CH2xe2x80x94Oxe2x80x94, CH2xe2x95x90C(R)xe2x80x94CH2OC(O)xe2x80x94, CH2xe2x95x90C(R)OC(O)xe2x80x94, CH2xe2x95x90C(R)Oxe2x80x94, and CH2xe2x95x90C(R) CH2OC(O)N(R1)xe2x80x94
wherein:
R is hydrogen or a C1-C4 alkyl group;
A is xe2x80x94Oxe2x80x94 or xe2x80x94NR1xe2x80x94 where R1 is hydrogen or a C1-C4 alkyl group or R1 is xe2x80x94B-X, B1Q1, B2Q2 or B3Q3 where B, B1, B2, B3, Q1, Q2 and Q3 and X are as defined above in the respective formulae I, II, III and IV and
K is a group xe2x80x94(CH2)pOC(O)xe2x80x94, xe2x80x94(CH2)pC(O)Oxe2x80x94, xe2x80x94(CH2)pOC(O)xe2x80x94, xe2x80x94(CH2)pNR2xe2x80x94, xe2x80x94(CH2)pNR2C(O)xe2x80x94, xe2x80x94(CH2)pC(O)NR2xe2x80x94, xe2x80x94(CH2)pNR2C(O)Oxe2x80x94, xe2x80x94(CH2)pOC(O) NR2xe2x80x94, xe2x80x94(CH2)pNR2C(O)NR2xe2x80x94, (in which the groups R2 are the same or different) xe2x80x94(CH2)pOxe2x80x94, xe2x80x94(CH2)pSO3 xe2x80x94, or, optionally in combination with B, a valence bond and p is from 1 to 12 and R2 is hydrogen or a C1-C4 alkyl group.
Preferably the ethylenically unsaturated groups of all monomers copolymerised together are either the acrylate type or are the styrene type, and, most preferably each has the same formula.
Preferably the zwitterionic monomer has the general formula VI 
wherein R, A and B are defined above,
the groups R3 are the same or different and each is hydrogen C1-1 alkyl, aryl, alkaryl, aralkyl, or two or three of the groups R1 with the nitrogen atom to which they are attached form a saturated or unsaturated hetero cyclic ring, and e is 1 to 6, preferably 2 to 4.
Terpolymers formed from the above mentioned zwitterionic monomer, a cationic comonomer of the formula II and a hydrophobic monomer of the formula III as well as some quaterpolymers are novel compounds and are claimed in our copending application filed even date herewith (agents ref HMJ02820WO).
By incorporating pendant groups to provide stable binding on the surface, the terpolymers can be stably bound to many types of underlying surface.
Instead of passing anticoagulant-treated blood through n extra corporeal filter, heparin (or other anticoagulant) scavenging may be carried out by implanting, permanently or temporarily, a device into the body in the circulation, which can remove anticoagulant which has been administered systemically. Thus the zwitterion and cation group carrying surface may be the surface of a vascular stent introduced into a blood vessel of a patient. In this embodiment the device may act as a reservoir, formed in situ, of active ingredient which may be released slowly into the circulation over an extended period of time. Alternatively a device may be preloaded with counterionically charged mucopolysaccharide or other active anionic compound, prior to implantation, to act as a slow release drug delivery system. In this system the anionic mucopolysaccharide or other anionic active compound is the counterion which is anion exchanged in the process of the invention.
The process of the present invention may include a subsequent step of recovering the anionic compound from the substrate by treating it with a second aqueous liquid containing a suitable anion regenerant which anion exchanges to displace the anionic compound.
The proportions of zwitterionic and cationic pendant groups in polymers used in the present processes and products depends upon the desired end use. Where high levels of mucopolysaccharide are to be scavenged from a fluid composition and/or it is desired for a high density of anionic mucopolysaccharide to be deposited onto a surface for subsequent use, then the amount of cationic pendant group should be relatively high as compared to the levels of zwitterionic groups. However where lower levels of mucopolysaccharide are required to be adsorbed to achieve anti-thrombogenic performance, whilst minimising deposition of protein and lipid components and platelets forms an important characteristic of the surfaces, then high levels of zwitterionic pendant groups are likely to be desirable. The relative ratios (equivalents) is in the range 1:100 to 100:1 (zwitterionic to ionic) preferably 1:10 to 10:1, more preferably 1:2 to 20:1.
Termonomer may be present in a monomer mix, for instance to provide the polymer with adsorption properties at a surface or covalent bonding to an underlying substrate, termonomers may be used. The total molar proportion of such termonomer in the polymer may be in the range 0.1 to 75%.
The copolymers and terpolymers may include diluent comonomer. Such diluent comonomer may be used in quantities up to 90 mol %, usually less than 50 mol %. Copolymerisable nonionic monomers may be used such as C1-24 alkyl(meth)acrylates, -(meth)acrylamides, and hydroxy C1-24alkyl(meth)acrylates and (meth)acrylamides.
The copolymers or terpolymers may include anionic pendant groups, to provide intermolecular crosslinking by counterionic bonding with cationic groups. In such cases, the equivalent level of anionic groups is lower than that of cationic groups in order that the polymer has an overall cationic charge. Anionic copolymerisable monomers may be used, for instance in which the anionic group is derived from carboxylic, sulphonic or phosphonic acid.
It has been found that the cationic/zwitterionic polymer is very stable and resistant to fouling during use.
The following examples illustrate the invention.
Performance Tests
Heparin Activity
Loading of samples with heparin
1. Filter Strips.
Samples were incubated with 5 ml of a solution of heparin in PBS (usually 50 U/ml. In other experiments, a heparin concentration of 4 or 200 U/ml in saline produced the same heparin surface activity on the cationic coated surfaces) for 30 min on a test tube shaker at room temperature. After 30 min, the samples were rinsed for 10 sec on both sides first with PBS then with deionized water. The samples were dried on tissue paper and in air and stored at room temperature.
2. Whole Filters.
Arterial filters were filled with 100 ml of a heparin solution in PBS (50 U/ml) and inlet/outlet sides were closed. The filter was rotated for 30 min, ensuring that all parts of the device were in contact with the heparin loading solution. The filter was then drained and filled/drained 3 times with PBS and then filled/drained 3 times with deionized water. The filter was dried by a stream of air and stored at room temperature.
Heparin loaded filter strips (dip-coated or removed from whole arterial filters) were usually incubated for 5 hrs at 37xc2x0 C. in PBS/BSA 1%/NaN3 0.1% to remove unstable bound heparin. The samples were then rinsed with PBS and deionized water as described and dried in air. Samples of 0.2-0.4xc3x970.4 cm were cut out and tested as described below.
A chromogenic assay (Heparin CRS106, Sigma). The xe2x80x9cSemi-Micro Methodxe2x80x9d described in the manual was used. Heparin loaded coated samples were placed in polystyrene test tubes. The tubes were placed into a 37xc2x0 C. water bath (5 tubes). 200 xcexcl of bovine factor Xa was added and the tubes were shaken. Following 1 min agitation, 200 xcexcl factor Xa substrate was added to the tubes and they were agitated for 5 min. 200 xcexcl acetic acid ( greater than 90%) was added to the tubes and the tubes were shaken. 200 xcexcl of the solution was removed from the tubes and added to the well of a microplate (2 wells/sample) and measured at 405 nm against wells containing 200 xcexcl of PBS. Previous results had shown that PBS gave the same absorbance reading as a reagent blank. The heparin activity was calculated with the use of a standard curve prepared with soluble heparin.
Heparin loaded and heparin free samples were incubated with human blood (citrate or heparin as anticoagulant) for 2-3 hrs and the degree of platelet adhesion was determined by scanning electron microscopy.
Samples of heparin loaded or heparin-free coated material were incubated with human plasma for 10 min, washed with PBS/BSA 1%, then incubated for 30 min with an anti-human fibrinogen antibody conjugated to horse radish peroxidase (Dako Code No. A080). The samples were washed and bound antibody was determined by incubating the samples with a substrate for peroxidase (O-phenylenediamine dihydrochloride, 0.4 mg/ml) and a phosphate citrate buffer with urea hydrogen peroxide (sigma P-9305). After 10 min the absorbance at 450 nm was measured against a reagent blank.
Two arterial filters (a control filter and a coated heparin loaded filter or a coated non-heparin loaded filter) were perfused in parallel for 6 hrs with bovine blood (3.5 L/min) at reduced heparin concentrations and macroscopic blood clots were detected visually and photographs were taken.
The counter ion in the polymeric system is chloride ion. Quantification of the chloride ion allows the level of cationic methacrylate to be determined.
Add 0.25 g polymer to 25 ml methanol. Once the material has fully dissolved add 75 ml of distilled water to the polymer/methanol mixture. Adjust the pH of the mixture to fall between 8-9. Add 1.0 ml of potassium chromate (5% in w/v distilled water) by pipette to the flask, and titrated to the first brown/red end-point with standardised 0.01 m silver nitrate solution. Repeat the titration, using 75 ml distilled water, but no polymer sample to obtain a blank reading. The level of cationic methacrylate in the polymer is directly proportional to the chloride ion concentration.